Retroviruses that do not carry transforming oncogenes can induce diseases by insertional mutagenesis of cellular loci, resulting in activation of protooncogenes or inactivation of tumor suppressor genes. Proviral integrations associated with gene mutagenesis can be identified by isolation of common integration sites, and retrovirus tagging of these sites through molecular cloning of viral-cellular junctions provides molecular access to the affected cellular genomic regions. A number of genes identified in this fashion have provided an entree into signaling pathways controlling cell growth, factor responsiveness, differentiation, homing, susceptibility to apoptosis and other functions. Our studies focus on the identification and characterization of novel cellular genes associated with retroviral insertional mutagenesis in lymphoma/leukemia. In our earlier work, we have cloned the Evi5 locus which is a common integration site of retroviruses in T-cell lymphomas of mice. We have further identified the Evi5 gene and demonstrated that it is a member of a well conserved novel multigene family. We have determined that the Evi5 gene is widely expressed in various normal mouse tissues and in some T-cell lymphomas the coding region of the Evi5 gene was truncated as a result of virus integrations. The objective of this study is to characterize the function of the Evi5 gene. The Evi5 gene encodes a 809 aa novel protein, which contains a 200 aa domain homologous to the human transforming gene tre. The mechanism of the tre oncogenicity remains elusive, despite that a role of the tre domain in protein-protein interaction has been implicated in recent studies. BLAST search indicated that, from yeast to human, several other genes contain high levels of overall similarity to the Evi5 gene on the protein level, while the function of these genes are unknown. We have found that RN-tre, a recently identified human gene exhibits limited overall similarity to the Evi5 (in addition to a tre homology). The RN-tre was identified through its ability to interact with the SH3 domain of eps8, a novel EGFR mitogenic transducer. Studies from others have shown that the RN-tre protein binds to eps8 in vivo with strong preference over several other SH3 domains of signal transducers. A C-terminal truncation mutant of RN-tre was able to confer growth advantage and reduce serum requirement to NIH3T3 cells, suggesting a role of RN-tre in regulation of cell growth. In this study, we explored the similarities between the Evi5 and RN-tre genes, and employed molecular genetic and biochemical approaches to use the eps8-binding activity as a tool to reveal a role of Evi5 in receptor tyrosine kinase signal transduction. Using genetically engineered GST-Evi5 fusion proteins, we have demonstrated that the N-terminus of the Evi5 protein binds to the eps8 protein in vitro, and determined that the Evi5 protein can form complex with eps8 and EGFR in vivo, in NIH3T3 and EGFR-transfected NIH3T3 (NIH-EGFR) cells. Further, we found that the Evi5 protein is constitutively phosphorylated on tyrosine in NIH-EGFR cells with amplified EGFR signals. We have transfected Evi5 expression vectors into NIH3T3 cells to study the biological function of Evi5. An Evi5 mutant of N-terminal deletion exhibited transforming activity resulting in foci formation of the transfectant cells. Coincidentally, the Evi5-eps8 binding region was deleted in this transforming mutant of Evi5. These studies indicated that Evi5 plays a role as a potential signal transducer of receptor tyrosine kinase pathway, and that disruptions of the Evi5 expression can lead to transformation of cells. Further characterization of the molecular mechanism of Evi5 transformation will be important to understand how Evi5 contributes to T-cell lymphomagenesis as well as to the normal cellular functions.